GSM7244928: Single cell RNAseq of FACS sorted VitA- Col1a1+  cells from DEN+WAD mouse liver.; Mus musculus; RNA-Seq
Liver non-parenhcymal cells were isoalted by in-situ liver perfusion with pronase and collagenase and vitamin A+ (VitA+) and VitA- Col1a1-expressing cells were collected via FACS on ARIA IIu (Becton Dickinson) with solid state laser excited at 488nm and measured with 510/20BP emission mirrors for GFP and excited at 355nm and measured with 450/50BP mirrors for VitA and data were analyzed by FlowJo 10.8.1 version.  The cells were immediately bar-coded for scRNA-seq. The library was prepared immediately after cell sorting with the target cell number of 10,000 and validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (Invitrogen) and qPCR (KAPA Biosystems) according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and  sample index were added.